Abstract
An increasing number of intracellular and extracellular proteins are shown to interact with membrane lipids under physiological conditions. For rapid and robust quantitative measurement of lipid-protein interaction, we developed a sensitive fluorescence quenching-based assay that is universally applicable to all proteins and lipids. The assay employs fluorescence protein (FP)-tagged proteins whose fluorescence emission intensity is decreased when they bind vesicles containing quenching lipids. This simple assay can be performed with a fluorescence plate reader or a spectrofluorometer and optimized for different proteins with various combinations of FPs and quenching lipids. The assay allows a rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid-binding proteins, and high-throughput screening of molecules that modulate their membrane binding.
Keywords:
Dark quenchers; Fluorescence proteins; High-throughput fluorescence assay; Lipid specificity; Membrane protein binding; Membrane-binding inhibitors.
© 2017 Elsevier Inc. All rights reserved.
MeSH terms
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Membrane / chemistry
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Cell Membrane / metabolism
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Cloning, Molecular
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Fluorescent Dyes / chemistry
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Gene Expression
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Glutathione Transferase / genetics
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Glutathione Transferase / metabolism
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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High-Throughput Screening Assays*
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Membrane Lipids / chemistry
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Membrane Lipids / metabolism*
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Phosphatidylcholines / chemistry
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Phosphatidylcholines / metabolism
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Phosphatidylinositol Phosphates / chemistry
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Phosphatidylinositol Phosphates / metabolism*
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Protein Binding
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Proto-Oncogene Proteins c-akt / genetics
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Proto-Oncogene Proteins c-akt / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism*
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Spectrometry, Fluorescence / methods*
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Unilamellar Liposomes / chemistry
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Unilamellar Liposomes / metabolism
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p-Dimethylaminoazobenzene / analogs & derivatives
Substances
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Carrier Proteins
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Fluorescent Dyes
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Membrane Lipids
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Phosphatidylcholines
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Phosphatidylinositol Phosphates
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Recombinant Fusion Proteins
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Unilamellar Liposomes
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enhanced green fluorescent protein
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phosphatidylinositol 3,4,5-triphosphate
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Green Fluorescent Proteins
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p-Dimethylaminoazobenzene
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Glutathione Transferase
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AKT1 protein, human
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Proto-Oncogene Proteins c-akt
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1-palmitoyl-2-oleoylphosphatidylcholine